Chemically programmable immunity

ABSTRACT

Methods and compositions for immediately immunizing an individual against any molecule or compound. The present invention comprises an immunity linker with at least two sites; ( 1 ) at least one first binding site that binds to an immune response component in an individual that has been pre-immunized with a universal immunogen, and ( 2 ) at least one second binding site that binds specifically to a desired compound or molecule, the target.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 10/969,770 filed Oct. 29, 2003, which is a continuation-in-partof U.S. application Ser. No. 10/178,046 filed Jun. 21, 2002, which is acontinuation of PCT/US00/35179 filed Dec. 21, 2000, which claims thepriority benefit of U.S. application Ser. No. 60/171,707 filed Dec. 22,1999.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods forproviding immediate immunity to any desired antigen. “Immunity” is usedherein to signify functional binding of immune components to a specifictarget. The specific target is not the same as, or structurally relatedto, the agent which generated the immunity in the first place, and infact, by the methods disclosed herein, the nature of the target isdetermined not by an immunogen alone, but also by a pharmaceuticalentity termed the “linker.” The “linker” connects an immune response,elicited by one entity, the universal immunogen, to another entity, thetarget, by means of two structural recognition sites on the linker. Thefirst of these sites binds to the recognition components of the immuneresponse. The second of these sites binds to the target. Accordingly,the term “immunity” is used in a way so as to include a process notpreviously known in immunology referred to here as the altermune method.The immunity conferred by the altermune method is dependent on classicalimmunity, and can be seen as an extension or diversion of it.

BACKGROUND OF THE INVENTION

[0003] Immunization has been used for over a hundred years to protecthumans and animals against disease. The premise of traditionalimmunization is that the most effective immune responses to an antigen,or a pathogen containing the antigen, occur after an individual ischallenged with that same antigen two or more times. This phenomena iscalled immunological memory or a secondary immune response. When theimmunization is successful, the individual is protected from the effectsof the pathogen from which the antigen was derived.

[0004] For example, once an individual is successfully immunized with anantigen derived from a bacterial organism, the immune system in thatindividual is primed and ready to respond to that bacteria when it isencountered. Successful immunization requires that the antigen islocated on an area of the bacteria that is accessible to theindividual's immune system. When successful, the immune system responds,the bacteria is killed, contained, neutralized, or otherwise clearedfrom the body, and little or no disease results from the infection bythe bacterial organism. The key to this protection is that immunizationwith the antigen must occur prior to the exposure to the bacterialorganism from which the antigen is derived.

[0005] Accordingly, the traditional immunization process generallyincludes injecting an antigen into an individual, waiting an appropriateamount of time, and allowing the individual to mount an immune response.The time required for mounting an immune response is betweenapproximately two weeks and several months for most antigens. In mostcases, a booster administration of the antigen is required to maintainthe immune response. This booster is normally given weeks or monthsafter the initial administration of the antigen.

[0006] Therefore, traditional immunization is highly successful atproviding protection if given several months in advance of exposure toan antigen, or pathogen, but traditional immunization is of little usewhen an individual is exposed to a new antigen to which the individualhas not been previously exposed and an immediate effective immuneresponse is required. A good example of such a situation is militarytroops in need of protection from bioterrorism agents. While apopulation of individuals can be vaccinated against agents ofbioterrorism in advance of any potential exposure to the agents,traditional vaccination is not a simple answer. Traditional vaccinationof a population creates harmful reactions in some persons and there ispotential that the population may never be exposed to the agent, yetrisks were taken. Additionally, a government cannot logisticallydevelop, produce and vaccinate essential personnel with vaccines forevery possible agent of bioterrorism. Accordingly, what is needed is acomposition that can be administered either immediately before, or evenafter, an individual's contact, or suspected contact, with a pathogen,which administration allows for the generation of an immediateprotective or effective immune response in the individual.

[0007] As alluded to above, another shortcoming of traditionalimmunization procedures is the requirement that the infectious pathogen,or a portion of the infectious pathogen, be administered to anindividual. There are numerous incidences wherein vaccinations havethemselves caused illness and even death because they contain a pathogenor a portion of a pathogen. Accordingly, what is needed is a compositionthat can be administered to an individual for immunization that does notcontain a portion of the pathogen against which the individual is beingimmunized.

[0008] Still another shortcoming of traditional immunization proceduresis the requirement that separate immunization procedures be used foreach antigen, although in some cases several antigens are included in asingle procedure. These separate immunization procedures are requiredbecause the natural memory, or secondary, immune responses are specificfor the antigen to which the primary immune response was directed.Accordingly, what is needed is a “universal immunogen” that can beadministered to an individual that will prime the individual's immunesystem for an immune response and a means to direct this immune responseto new targets as the need arises. Alternatively, there is a need forthe means to re-direct an existing immune response to a new target. Sucha “universal immunogen,” or the means of re-directing an existing immuneresponse, would reduce the number of immunizations currently recommendedfor individuals.

SUMMARY OF THE INVENTION

[0009] The present invention provides compositions and methods for aprogrammable immunity that can provide a substantially immediate immuneresponse by an individual against a target, such as a pathogen or otherundesired substance. Since an immediate effective immune response isachieved, these compositions may be administered to an individual anytime prior to the individual's contact with a pathogen or even soonafter the individual's contact with a pathogen. In one embodiment, thepresent invention solves many of the problems facing the militaryregarding protection of their personnel from agents of bioterrorism.

[0010] The compositions and methods of the present invention alsoprovide an advantage over traditional immunization techniques becausethe methods do not require that a modified pathogen or a portion of apathogen be administered to an individual for effective immunizationagainst that pathogen. Accordingly, the present invention will save thelives of people that currently have fatal adverse reactions totraditional vaccines.

[0011] The compositions of the present invention, in one embodiment,include an immunity linker, containing at least one first binding sitethat binds to an immune response component; and containing at least onesecond binding site that binds to a target. These linker compositionsmake use of a pre-existing immune response in an individual and linkthat pre-existing immune response to a different target, which isunrelated to the pre-existing immune response except for the connectionbetween the two provided by the immunity linker. The pre-existing immuneresponse is directed to an antigen containing the first binding site ofthe immunity linker and can be induced in the individual byadministration of a universal immunogen containing the first bindingsite. Linking the immune response to the target allows for an immediate,linked immune response without the requirement for a primary immuneresponse against the target.

[0012] The immunity linker can be any type of chemical or biologicalmaterial including a microbe, a bacteriophage, a protein, a nucleicacid, a polysaccharide, a synthetic material or a combination thereof.In one embodiment, the at least one first binding site is physically orchemically linked or conjugated to a molecule comprising the at leastone second binding site. In this embodiment, a spacer molecule mayreside between the first binding site and the second binding site. Inanother embodiment, the immunity linker is a single molecule containingthe at least one first binding site and the at least one second bindingsite.

[0013] A universal immunogen is administered to an individual andcorresponds to, or contains, the first binding site of the immunitylinker. Such administration causes the individual to mount a cellularand/or a humoral immune response, depending on the immunogen and theroute of administration. If necessary, the universal immunogen can beadministered with an adjuvant or other immune response enhancingmaterials. Additionally one or more booster administrations of theuniversal immunogen may be given to the individual at appropriate times.These methods of immunizing an individual are well known to one ofordinary skill in the art. The pre-existing immune response,alternatively, can be an immune response that does not requireadministration of a universal immunogen such as, but not limited to, thealpha-Gal response universal in humans as described herein.

[0014] Following the initiation or generation of a pre-existing immuneresponse, if the individual is exposed, or is suspected of beingexposed, to a novel compound or pathogen for which immediate immunity isdesired, the individual is administered an immunity linker describedherein that contains a first binding site that corresponds to theuniversal immunogen and a second binding site that binds to the novelcompound or pathogen. The immunity linker binds at the one first bindingsite to the immune response components produced during the pre-existingimmune response, and also binds to the novel compound or pathogen at theat least one second binding site thereby providing an immune complex ofthe immune response component bound to the immunity linker which is alsobound to the novel compound or pathogen. The immune system of theindividual recognizes these immunity linker complexes and removes orclears them from the body.

[0015] Thus, by administering a composition comprising an immunitylinker described herein, the pre-existing immune response of theindividual is re-directed from the universal immunogen to the novelcompound or pathogen. As mentioned above, another benefit of the presentinvention is that only one initial immunizing molecule or universalimmunogen is required for priming an individual's immune system for alater antigen-specific immune response. Thus, the present invention maydecrease the number (and possibly the complexity of formulation) ofvaccinations currently recommended or required for individuals. Afurther benefit of the present invention is the ease of preparation ofthe immunity linker and the universal immunogen. The immunity linkers ofthe present invention can be easily assembled and provided to healthcare professionals for rapid response to such public health needs aspandemic infections, bioterroristic threats, or limited outbreaks ofspecific pathogens. A still further benefit of the present invention isthe breadth of compounds against which an individual can be immunized.Immunity linkers can be created that bind to any compound or foreignmaterial such as antigens, pathogens, chemicals, or endogenous materialssuch as altered cells found in viral infections or cancer.

[0016] As previously mentioned, it is to be understood that the presentinvention can utilize an existing immune response in an individual.Thus, if an individual is already immune to a particular antigen, animmunity linker molecule can be made that has a first binding sitecomprising or corresponding to the antigen to which the individual isimmune and the second binding site can be directed to the undesiredpathogen, chemical or agent.

[0017] It should also be understood that the present invention allowsfor immune tuning, or in other words, allows for the selection of thetype of immune response generated by the universal immunogen and/or theimmunity linker, and thus allows for the selection of the type of immuneresponse directed toward any particular target. Accordingly, a firstbinding site of a universal immunogen and/or an immunity linker may bechosen based upon whether the first binding site elicits a humoralimmune response, a cellular immune response and/or an innate immuneresponse.

[0018] These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is related to methods and compositions forimmediately immunizing a human or animal against any molecule ororganism, referred to herein as a target. This is referred to aschemically programmable immunity or programmable immunity. Programmableimmunity differs from classical immunity in that programmable immunityallows for the re-direction of a pre-existing immune response directedtoward one antigen, to the target. The immune response is re-directedusing an immunity linker of the present invention.

[0020] As used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlyindicates otherwise. Thus, for example, reference to a “compound” is areference to one or more such compounds and includes equivalents thereofknown to those skilled in the art, and so forth.

[0021] Immunity linkers of the present invention comprise at least twosites; (1) a first binding site that binds to at least one immuneresponse component of an individual, and (2) a second binding site thatbinds to a target. The immune response component is one that exists inthe individual prior to administration of the immunity linker to theindividual. For example, the immune response component can be anantibody that was part of a previous immune response to the firstbinding site, or to a molecule, or a large assembly of molecules, oreven a micro-organism containing the first binding site. Accordingly, asused herein, the term “pre-existing immune response” refers to an immuneresponse that is directed toward the first binding site or an epitopethat is immunologically similar to the first binding site. In otherwords, a “pre-existing immune response” is an immune response in whichimmune response components are generated or exist that bind to the firstbinding site. The pre-existing immune response can be generated by aprevious administration to the individual of a universal immunogen thatcorresponds to a first binding site or can exist in the individualwithout such administration.

[0022] Accordingly, the present invention includes a method of divertinga pre-existing immune response in an individual from a first antigen toa target comprising, administering to the individual an effective amountof a composition comprising one or more immunity linkers, wherein thelinkers comprise at least one first binding site and at least one secondbinding site, wherein the second binding site binds to the target andwherein the first antigen comprises the first binding site or animmunological equivalent thereof. The present invention also encompassesa method of increasing an immune response to a target comprising,administering to the individual an effective amount of a compositioncomprising one or more immunity linkers, wherein the linkers comprise atleast one first binding site and at least one second binding site,wherein the second binding site binds to the target and wherein theindividual has a pre-existing immune response to the first binding siteor an immunological equivalent thereof.

Universal Immunogens

[0023] A universal immunogen that “corresponds to” a first binding sitecan be identical to the first binding site, can contain the entire firstbinding site, can contain a portion of the first binding site, or can bean immunological equivalent of the first binding site. When referring totwo or more molecules, the term “immunological equivalent” refers hereinto molecules that are bound by the same immune response component. Thepresent invention only requires that the immune response componentraised by the universal immunogen also bind to the first binding site.In one embodiment, the universal immunogen binds to the immune responsecomponent with sufficient affinity to result in the production of acomplex that is capable of initiating or participating in an immuneresponse. In a preferred embodiment, the cross-reactivity of the immuneresponse component to molecules other than the universal immunogen andthe first binding site is minimal.

[0024] The universal immunogen can be any molecule, organism or compoundto which an individual mounts an immune response and can be administeredvia any route. The universal immunogen can be, but is not limited to, amolecule, a microbe, or a toxin or a toxoid derived therefrom; a proteinor polypeptide; a polynucleotide; a polysaccharide; a synthetic materialor a combination thereof. Preferably the universal immunogen causes animmune response in an individual that provides for long-lasting immunememory, can be re-administered to individuals in booster doses, and doesnot cause disease, pathology or long-term illness in individuals. Animmunogen that comprises a portion of a pathogen or a modified portionof a pathogen can be a universal immunogen, but a universal immunogen isnot required to bear any relationship to anything except thecomplementary immune response which it elicits. For example, humans areroutinely immunized with immunogenic antigens from mumps virus, measlesvirus, tetanus toxoid, and poliovirus. Animals, such as cats and dogs,are routinely immunized with immunogenic antigens from rabies virus.These and other traditional immunogens can be used as universalimmunogens, however, this would be a matter of convenience, notnecessity.

[0025] Alternatively, non-traditional immunogens may be used as theuniversal immunogen. Preferably, a non-traditional immunogen does notcontain either a portion or a modified portion of a pathogen. In oneembodiment, the universal immunogen is a protein, or a portion of aprotein, to which a hapten is bound. A “hapten” is defined herein as amolecule that reacts with a specific antibody but cannot induce theformation or generation of additional antibodies unless bound to acarrier protein or other large antigenic molecule. Most haptens aresmall molecules, but some macromolecules can also function as haptens.In one embodiment, performed for demonstration purposes and describedherein as Example 1, the hapten is a phenylarsonate and the universalimmunogen is a phenylarsonylated protein.

[0026] In another embodiment, the universal immunogen comprises abacteriophage or an epitope of a bacteriophage. An immune responsecomponent can bind to any part of the bacteriophage and in oneembodiment, binds to a peptide that is expressed on the surface of thebacteriophage. A bacteriophage universal immunogen can be administeredto an individual via any route and in some embodiments, thebacteriophage can be contained within a bacteria as a convenient meansof administration.

First and Second Binding Sites and Spacers of an Immunity Linker

[0027] The present invention is able to re-direct a pre-existing immuneresponse directed toward a universal immunogen to a different antigen,in part, because the universal immunogen corresponds to a first bindingsite of an immunity linker. Since the first binding site is a part ofboth the universal immunogen and the immunity linker molecule, thepre-existing immune response, or the pre-existing immune systemcomponents, that are directed to the universal immunogen also recognizethe immunity linker. The first binding site of the immunity linker cancomprise a polypeptide, a polynucleotide, a polysaccharide, an organicchemical, a microorganism such as a bacteriophage, a bacterium, a virusor viral particle, or a protozoa, any fragment or portion of theforegoing, any combination of the foregoing, or any other compositionthat is recognized by the immune system of an individual or bound by animmune response component in an individual.

[0028] In one embodiment, the first binding site is the alpha-Galepitope, i.e.,galactosyl-alpha-1,3-galactosyl-beta-1,4-N-acetylglucosamine. In anotherembodiment, the first binding site comprises a portion of abacteriophage, and more preferably, a polypeptide that is expressed onthe surface of a bacteriophage.

[0029] The second binding site can comprise a polypeptide, apolynucleotide, a polysaccharide, an organic chemical, a microorganismsuch as a bacteriophage, a bacterium, a virus, a protozoa, or anyfragment or portion of the foregoing, any combination of the foregoing,or any other composition that binds to a target. In one particularembodiment the first binding site and the second binding site arecontained on the same microorganism. As used herein, polynucleotide ornucleic acid means either DNA or RNA, in any stranded conformation,e.g., single, double, triple, and any chemical modifications thereof,and contemplates the presence or absence of proteins associated with thenucleic acid. Chemical modifications can be in individual nucleotidesprior to amplification or synthesis of the nucleic acids, or can beadded to nucleotides after incorporation into multimers. Suchmodifications include, but are not limited to, modifications atcytosine, exocyclic amines, substitution of 5-bromo-uracil, backbonemodifications, methylations, unusual base-pairing combinations andothers known to those skilled in the art. In one embodiment, the secondbinding site comprises an antibody or an antibody fragment, preferablyan antibody fragment containing an antibody variable region, and morepreferably a Fab fragment. In another embodiment, the second bindingsite comprises a polypeptide expressed by a bacteriophage, and morepreferably, a polypeptide that is expressed on the surface of abacteriophage.

[0030] As described above, the immunity linker comprises any type ofmolecule or organism that contains a first binding site capable ofbinding to an immune response component, and contains a second bindingsite capable of binding a target. It is to be understood that theimmunity linkers can contain more than one first binding site and/ormore than one second binding site. The multiple first binding sites canbe identical or can be different. The multiple second binding sites canalso be identical or different. Binding sites may differ in theirspecificity for different molecules or their affinity for the samemolecule. The immunity linker can also be modified to reduce its ownimmunogenicity.

[0031] Binding by the first and second binding sites to the immuneresponse component and target, respectively, can be accomplished throughany interaction including through binding provided by other molecules,such as polysaccharides or nucleic acids. In a preferred embodiment, afirst binding site is specific for an immune response molecule and asecond binding site is specific for a target. As described above, amolecule is “specific for” another molecule when the two molecules bindwith sufficient affinity to result in the production of a functionalcomplex for purposes of the immune system. In a further preferredembodiment, the cross-reactivity of one second binding site withmolecules other than a target is minimal. In another preferredembodiment, the cross-reactivity of one first binding site withmolecules other than an immune response component is minimal.

[0032] Following administration of the immunity linker to theindividual, an immunity linker complex comprising the immune responsecomponent, the immunity linker, and the target is formed. The immunitylinker can bind the target prior or subsequent to the binding of theimmunity linker to an immune system component. Following formation ofthe immunity linker complex, the target is cleared via immune systempathways. A “clearing” of an antigen refers herein to the removal,inactivation or modification of the antigen such that it is no longerharmful to the body.

[0033] In another embodiment, the immunity linker comprises a firstbinding site that corresponds to an alpha-galactosyl, or alpha-Gal,epitope as is described in Galili, U. and Avila, J. L., Alpha-Gal andAnti-Gal, Subcellular Biochemistry, Vol. 32, 1999. Xenotransplantationstudies have determined that humans mount an immune response to thealpha-galactosyl epitope, which is not normally found in humans, but isfound in other animals and many microorganisms. In one particularembodiment of the present invention, the alpha-galactosyl epitope isconjugated to a second binding site that comprises an Fab fragment of anantibody.

[0034] In yet another embodiment, the immunity linker comprises abacteriophage. The first binding site can correspond to any portion ofthe bacteriophage, but preferably corresponds to a first polypeptideexpressed by the bacteriophage. A second binding site on thebacteriophage corresponds to a second and differentbacteriophage-expressed polypeptide that binds to a target. Both thefirst and second polypeptides are expressed on the surface of thebacteriophage.

[0035] Example 3 below provides one example of a bacteriophage immunitylinker as can be used in conjunction with a bacteriophage universalimmunogen. In some embodiments, the immunity linker comprises arecombinant bacteriophage derived from a wild type bacteriophage by themethod of phage panning. The first binding site consists of whateverportion (s) of the wild type bacteriophage function(s) immunogenicallyon the initial exposure of the subject to the wild type bacteriophageand is (are) still retained by the recombinant bacteriophage, which isused as a linker. The second binding site on the recombinantbacteriophage, used as a linker corresponds to a recombinantbacteriophage-expressed peptide that is selected because it binds to atarget.

[0036] In another embodiment the immunity linker is a conjugate of thealpha-Gal epitope, acting as a first binding site, and a syntheticpeptide, acting as a second binding site, the sequence of which peptideis derived from a bacteriophage panning experiment, wherein the peptideis panned from a random peptide library, displayed on a collection ofrecombinant bacteriophage, by the intended antigen attached to a solidsupport.

[0037] In other embodiments, the first and/or second binding sitescomprise an aptamer nucleic acid, and more preferably an aptamer thathas been produced by the SELEX process. SELEX stands for SystemicEvolution of Ligands by EXponential enrichment. SELEX methods are knownin the art and are described in at least the following issued U.S.Patents: U.S. Pat. Nos. 5,475,096; 6,261,774; 6,395,888; 6,387,635;6,387,620; 6,376,474; 6,346,611; 6,344,321; 6,344,318; 6,331,398;6,331,394; 6,329,145; 6,300,074; 6,280,943; 6,280,943; 6,280,932;6,261,783; and 6,232,071.

[0038] In general, the SELEX method relates to identifying nucleic acidsthat specifically bind to three dimensional targets. Nucleic acids havesufficient capacity for forming a variety of two- and three-dimensionalstructures and sufficient chemical versatility available within theirmonomers such that some sequences can be found that bind specificallywith virtually any chemical compound. For purposes of stability inbiological fluids, a preferred aptamer contains one or more modifiednucleotides such as 2′-fluoro- or 2′-amino-2′-deoxypyrimidines. Nucleicacids using these bases are much more stable in vivo than naturallyoccurring nucleic acids. See, M. Famulok and G. Mayer, Cur. Top. Micro.Immunobiol. 243:123-146, 1999. Spiegelmers (see Vater, A. and Klussmann,S. Current Opin. Drug Discov. Devel. 2003 Mar; 6(2):253-61) derived bysimilar methods may also be employed for their inherent stability inserum.

[0039] The first and second binding sites of the immunity linkerdescribed herein may be linked, or conjugated, by any means known to oneof skill in the art. The terms “conjugated” and “conjugation” aredefined herein to refer to a covalent or other form of linking two ormore molecules. Conjugation can be accomplished by any means including,but not limited to, chemical means, genetic engineering means, or invivo by biologic means. The first and second binding sites may be linkedby a double stranded nucleic acid, a polypeptide, a chemical structure,or any other appropriate structure, or may be linked by a simplechemical bond.

[0040] In one particular embodiment the first and second binding sitesof a linker are evolved in vitro in such as way that the first bindingsite will only interact with the immune response component after thesecond binding site has bound to the target. Allosteric interactionsleading to such behavior are well-known in proteins and othermacromolecules, and could be a component of the selection process in thein vitro evolution of the linker. The linker may also be made bynanofabrication methods.

Immune Response Components

[0041] As stated above, the one or more first binding sites of theimmunity linker bind to an immune response component. The term “immuneresponse component” is used herein to refer to any molecule or cellinvolved in an immune response of an individual. The term “individual”encompasses both animals and humans. Non-limiting examples of immuneresponse components are antibodies; lymphocytes including, but notlimited to, T cells, B cells and natural killer cells; macrophages;granulocytes including, but not limited to, neutrophils, basophils andeosinophils; and receptors on any of the foregoing cells including, butnot limited to, T cell receptors and B cell receptors. A humoral immuneresponse component includes an antibody. The term antibody includes allof the classes and subclasses of antibodies, IgG, IgM, IgA, IgD, IgE,etc., secretory and excreted forms of the antibodies, fragments ofantibodies, including variable, hypervariable and constant regions,heavy and light chains, combinations of fragments and mixtures offragments and whole antibodies. Such antibodies can be humanized,polyclonal or monoclonal, naturally derived or synthetic antibodies.

[0042] In one embodiment, at least one first binding site binds to theactive binding site of the immune response component. For example, ifthe immune response component is an antibody such as an IgG molecule,the first binding site of the immunity linker is the antigenic epitopeto which the active binding site of the variable region of the IgGmolecule normally binds.

Targets

[0043] The one or more second binding sites of the immunity linkers bindto a target, and preferably the second binding site is specific for thetarget. The term “target” refers herein to any composition to which anincreased immune response is desired in an individual.

[0044] In one embodiment, the antigen is a compound or organism to whichthe subject individual has not been exposed. However, the antigen mayalso be a compound or organism, to which the subject individual has beenexposed but to which an optimal immune response has not been mounted.

[0045] Targets include, but are not limited to, antigens,microorganisms, pathogens, viruses, viral particles, bacteria,polypeptides, toxic chemicals, non-self molecules, and any fragments,portions or combinations thereof. As used herein, targets also includemolecules or compositions that are not normally targeted by an immuneresponse in an individual, such as molecules identifiable as self,molecules too small to be responded to by the immune system,nonimmunogenic compounds or chemicals, and molecules or materials thatare sequestered from the immunogenic components of the immune system. Inone embodiment, the target is an antibody or cellular component of theimmune system that the individual wants to eliminate so as to relieve anautoimmune disorder.

Immunity Linker Populations

[0046] As indicated above, the immunity linkers of the present inventioncan have more than one first binding site and/or more than one secondbinding site. The present invention also encompasses the use of one ormore populations of immunity linkers wherein each population has adifferent first binding site and/or second binding site. The multiplebinding sites may differ either in their specificity for differentmolecules or epitopes or their affinity for the same molecule orepitope. In one embodiment of the present invention, the immunity linkercomprises two or more second binding sites, each specific for adifferent target. In another embodiment, the immunity linker comprisestwo or more second binding sites, each specific for different epitopeson the same target. In yet another embodiment, the immunity linkercomprises two or more second binding sites, each specific for the sameepitope on a target but having different affinities for the target.

[0047] In still other or further embodiments, the immunity linkercomprises two or more first binding sites, each capable of binding to adifferent immune response component. In yet another embodiment, theimmunity linker comprises two or more first binding sites, each capableof binding to different sub-structures of the same immune responsecomponent. In another embodiment, the immunity linker comprises two ormore first binding sites, each capable of binding to the samesub-structure of an immune response component but having differentaffinities for the immune response component.

[0048] The immunity linkers of the present invention can have anycombination of the aforementioned multiple first binding sites andsecond binding sites. The present invention also encompasses theadministration of different populations of immunity linkers, eachpopulation having any combination of the aforementioned multiple firstbinding sites and second binding sites.

[0049] In one embodiment, a population of immunity linkers isadministered to an individual, wherein each linker has an identicalfirst binding site and the second binding sites are all aptamers, thatbind to the same target, but with different affinities for the target.In another embodiment, a population of immunity linkers is administeredto an individual, wherein each linker has an identical first bindingsite and the second binding sites are all antibodies, or portions ofantibodies, that bind to different targets. In still other embodiments,the immunity linkers of the population all have an identical firstbinding site and different types of second binding sites, i.e., antibodybinding sites, aptamer binding sites, etc., where each second bindingsite is specific for the same target or different targets.

[0050] The present invention contemplates populations of immunitylinkers that comprise at least one first binding site described herein.Such populations can have immunity linkers all having first bindingsites having the same binding specificity or combinations of bindingspecificities. Further, the binding may be accomplished by first bindingsites of the same type, such as all being nucleic acid molecules or allproteins, which may have the same or different binding specificities.The binding may be accomplished by first binding sites of differenttypes on one immunity linker or a population of different immunitylinkers with differing first binding sites. The first binding sites ofdifferent types can have the same or different binding specificities forone or more immune response components.

[0051] Additionally, the present invention contemplates populations ofimmunity linkers that comprise at least one second binding sitedescribed herein. Such compositions comprise immunity linkers all havingsecond binding sites having the same binding specificity or combinationsof binding specificities. Further, the binding may be accomplished bysecond binding sites of the same type, such as all being nucleic acidmolecules or all proteins, which may have the same or different bindingspecificities. The binding may be accomplished by second binding sitesof different types on one immunity linker or a population of differentimmunity linkers with differing second binding sites. The second bindingsites of different types can have the same or different bindingspecificities for one or more targets.

[0052] Thus, the compositions comprise immunity linkers in which thebinding specificity of the at least one first binding site and thebinding specificity of the at least one second binding sites are alluniform, that is, each first binding site has the same bindingspecificity for its binding partner and each second binding site has thesame binding specificity for its binding partner. Alternatively thecompositions may comprise multiple immunity linker populations eachpopulation having first binding sites with differing bindingspecificities and also having second binding sites with differingbinding specificities.

Methods of Use

[0053] The present invention comprises methods and compositions fordiverting a pre-existing immune response in an individual from a firsttarget to a second target. In some embodiments, both the first targetand the second target are different antigens. Since the first antigen,or an immunological equivalent of the first antigen, is present in thelinker molecule, the “diverting” of an immune response does not requirea cessation of the immune response to the first antigen. The presentinvention further provides methods and compositions for increasing animmune response to a target in an individual. A previous immune responseto the target may or may not already exist in the individual. Thepresent invention also provides chemically programmable immunity forindividuals that provide for the immediate and specific immunization ofthe individual against a pathogen or other undesired substance.

[0054] According to the present invention, the individual is firstimmunized with a universal immunogen. The individual can then beimmediately immunized against a chosen target simply by administering tothe individual a composition comprising an immunity linker with at leastone first binding site that binds to an immune response component and asecond binding site that binds to a target. Any combination of universalimmunogen and immunity linker described herein can be used with the onlyrequirement that the first binding site of the immunity linker will bebound by some of the immune response components produced as a result ofinoculation by the universal immunogen. Immunity to the universalimmunogen may occur as a result of an intentional inoculation or, as inthe case of the alpha-Gal epitope and its attendant anti-Gal immunity,by natural processes.

[0055] The present invention may be particularly useful in the militarywhere troops may be unexpectedly exposed to a pathogen, toxin, or to atoxic chemical substance. Military personnel are pre-immunized with auniversal immunogen that corresponds to the first binding site of animmunity linker. If the military personnel are unexpectedly challengedor believed to be challenged with a pathogen, toxin, or chemical agent,the immunity linker, having a second binding site that binds thepathogen, toxin, or chemical agent, is administered to the militarypersonnel, thereby immediately protecting them against the pathogen.

[0056] The present invention can be used to prevent and/or treat diseaseor infection from organisms including, but not limited to, anthrax,dengue virus, and Marburg virus. For example, upon detecting anthrax ina combat zone, immunity linkers specific for anthrax are administeredorally to the troops and civilians previously immunized with theuniversal immunogen and protection against anthrax is conferred. Theimmunity lasts as long as the personnel continue to maintain adequate invivo concentrations of immunity linkers. In one embodiment, immunitylinkers are administered to the individuals on a continuing basis inorder to maintain adequate in vivo concentrations of immunity linkers.Immunity linkers can be administered at any interval including, but notlimited to, hourly, daily, weekly, or monthly intervals. In the case ofimmunity linkers that must necessarily be administered for a long periodof time, linkers are sought wherein the second binding site is notitself immunogenic. Once the threat is passed, administration ofimmunity linkers is stopped. Possible side effects of the presentinvention are therefore temporary, unlike traditional immunizationswhich often generate long-lasting side effects or complications inimmunized humans or animals.

[0057] With regard to the more general population, pharmacies can have alibrary of different immunity linkers available for a variety ofdifferent pathogens and toxic substances. Once an individual ispre-immunized with a universal immunogen, administration of one or moreof these different immunity linkers results in the generation of aprotective immune response against the variety of different pathogensand toxic substances.

[0058] One example of an infection treatable by the present invention isthe flu or infection by an influenza virus. By using the immunitylinkers of the present invention, there is no need to develop a newstrain of vaccine every year to respond to the new strain of influenzaof that year. Only one portion of the immunity linker needs to bealtered as the influenza virus alters its antigenic markers. The atleast one second binding site can be changed each year, or as needed, tothat which binds to the new influenza virus of that occurrence.Preferably, the at least one second binding site is a DNA aptamer madefrom modified nucleotides. Such DNA molecules are very stable againstmetabolic enzymes. For example, individuals can inhale compositions ofimmunity linkers having the appropriate first and second binding sitesto prevent the attachment and infection by influenza virus. Thisinhalation therapy continues as long as necessary and is stopped whenthe influenza season has passed.

[0059] It should be understood that the present invention also allowsfor immune tuning, or in other words, allows for the selection of thetype of immune response generated by the universal immunogen and/or theimmunity linker, and thus allows for the selection of the type of immuneresponse directed toward any particular target. A first binding site ofa universal immunogen and/or an immunity linker may be chosen based uponwhether the first binding site elicits a humoral immune response, acellular immune response and/or an innate immune response. A humoralimmune response is defined herein as an immune response mediatedpredominantly by antibodies. A cellular immune response includes animmune response mediated predominantly by T cells. A first binding sitemay also be chosen based upon whether it elicits a CD4⁺ T cell responseor a CD8⁺ T cell response. As used herein, a CD4⁺ T cell response is animmune response mediated predominantly by CD4⁺ T cells as compared toCD8⁺ T cells, whereas a CD8⁺ T cell response refers herein to an immuneresponse mediated predominantly by CD8⁺ T cells as compared to CD4⁺ Tcells. An innate immune response includes an immune response mediatedpredominantly by macrophages and/or NK cells. Accordingly, the presentinvention includes immunity linkers and universal immunogens thatcontain a first binding site that elicits a humoral immune response, acellular immune response, an innate immune response, a CD4⁺ T cellimmune response and/or CD8⁺ T cell immune response.

[0060] The ability to tailor the immune response generated by theuniversal immunogen and/or the immunity linker is important. In somediseases, chiamydia infections of the eye, for instance, it is thoughtby some that the immune response is the cause of the pathology. Havingcontrol of the nature of the immune response independent of the natureof the pathogen may have an important role in treatment. For instance,it may be the cellular component of the immune response to chlamydiathat leads to blindness; if so, the altermune therapy of the presentinvention for chlamydia could be based on an immunity, which does notinvoke a cellular immune component, killing the bacteria through a Tcell-independent mechanism. Thus unlike the natural immune system, andfor the first time, the profile of an immune response can be tailored bya physician using the present invention and not pre-determined by thenature of the pathogen itself.

[0061] Accordingly, the present invention includes a method ofincreasing a humoral immune response, a cellular immune response, a CD4⁺T cell immune response, and/or a CD8⁺ T cell immune response in anindividual to which a universal immunogen and/or an immunity linker areadministered. These immune responses may or may not be the type ofimmune response that would occur naturally in an individual in responseto a particular target. In some embodiments, the immune response isdifferent from that which would or does occur naturally in an individualin response to a particular target. In one embodiment, a universalimmunogen and an immunity linker are administered to an individual thatcontain a first binding site that elicits a humoral immune response,wherein the second binding site of the immunity linker binds to a targetthat usually elicits a cellular immune response in that or otherindividuals.

[0062] The present invention further comprises methods for removingother unwanted materials from the body of a human or animal byadministering a composition comprising an immunity linker. The immunitylinkers can be used to remove excess or unwanted molecules or chemicalssynthesized by the body or found in the body, including but not limitedto, proteins, fats, nucleic acid polymers, hormones, cellular factors,neurochemicals, toxic cellular factors, apoptotic factors, cellularsignal molecules, antibodies or unwanted cells, minerals such as calciumor magnesium and compounds comprising combinations or mixtures of theseand other molecules. It is contemplated that in some cases, complexmethods might be employed to remove unwanted cells such as marking themin such a way as to make them susceptible to immunity linker binding.The immunity linkers can be used to remove any unwanted material fromthe body by providing a second binding site that binds the unwantedmaterial and using the first binding site's binding to an immuneresponse component such that the body's natural clearance mechanisms areenlisted to remove the unwanted material. Any material that can be boundby the second binding site can be effected or removed by the methods ofthe present invention, thus the list of materials that can be effectedor removed is only limited by the ability to provide a binding partnerfor the unwanted material. Providing binding partners for unwantedmaterials is well within the scope of skilled practitioners and includesboth the methods discussed herein and others used by those skilled inthe art.

[0063] With the methods and compositions of chemically programmableimmunity, an immune response can be used to clear or contain theseunwanted materials such as if an immune response had been elicited bythe unwanted material directly. For example, antibody complexes,comprising immunity linkers, bound at one site to antibodies and atanother site to the unwanted material, are removed by the body's immuneclearance mechanisms. Containment of the target can comprise mechanismssuch as those wherein cells wall-off or form barriers around theimmunity linker bound to the unwanted material, similar to the cellularresponse used to wall off tuberculosis pathogens. In some methods,artificial mechanisms such as plasmaphoresis methods, wherein the bloodor other fluids are filtered outside of the body, can be used to entrapthe immune complexes or cellular complexes formed with immunity linkers.Specific removal of bound immunity linkers can be used, for example byusing columns or separation systems using antibodies to the immunitylinker itself.

[0064] Accordingly, the present invention may be used for the treatmentof multiple infections, diseases and conditions. The terms “treatment,”“treating,” “treat,” and the like are used herein to refer generally toobtaining a desired pharmacological and/or physiological effect. Theeffect may be prophylactic in terms of completely or partiallytransferring immunity from one antigen to another and/or may betherapeutic in terms of a partial or complete stabilization or cure fora disease and/or adverse effect attributable to the disease. “Treatment”as used herein covers using the immune response directed to one antigenfor the control of another antigen or its effects such as any treatmentof a disease in a subject, particularly a human, and includes: (a)preventing the disease or symptom from occurring in a subject which maybe predisposed to the disease or symptom, but has not yet been diagnosedas having it; (b) inhibiting the disease symptom, i.e., arresting itsdevelopment; or (c) relieving the disease symptom, i.e., causingregression of the disease or symptom. The terms “treatment,” “treating,”“treat,” and the like also include the reduction, control or containmentof an unwanted substance, including an antigen, in an individual. Theamount of reduction of a substance may be determined by any method.

[0065] The expression “therapeutically effective amount” refers to anamount of, for example, a composition disclosed herein, that iseffective for preventing, ameliorating, treating or delaying the onsetof a disease or condition. A “prophylactically effective amount” refersto an amount of, for example, a composition disclosed herein that iseffective for preventing a disease or condition.

Methods of Administration

[0066] According to the present invention, a universal immunogen isadministered to an individual prior to administration of a correspondingimmunity linker. A universal immunogen can be administered at any timeprior to administration of a corresponding immunity linker and may beadministered multiple times prior to administration of a correspondingimmunity linker. These multiple administrations may be referred to as“booster” administrations. One method contemplated by the presentinvention comprises multiple administrations of different universalimmunogens. With administrations of different universal immunogens, therepertoire of possible immune linkers is increased.

[0067] Multiple administrations of immunity linkers are also included inthe present invention. Methods include immunization of an individualusing one universal immunogen followed by one or more administrations ofthe same or different immunity linkers. Methods also includeimmunization of an individual using several different universalimmunogens followed by one or more administrations of the same ordifferent immunity linkers.

[0068] It is preferred that immunity linkers are administered to anindividual for as long as is needed and at appropriate intervals tomaintain adequate in vivo concentrations of the immunity linkers totreat an infection or disease or to remove sufficient amounts of anunwanted material from the individual. Immunity linkers can beadministered at any interval including, but not limited to, hourly,daily, weekly, or monthly intervals, or any division thereof.Appropriate administration intervals can be determined by those ofordinary skill in the art and are based on the identity of the target orpathogen, the amount of target or pathogen detected in the individual,duration of exposure, immune linker pharmacokinetics, characteristics ofthe individual such as age, weight, gender, etc., and any other relevantfactors. The time of administration of immunity linker will need to beempirically determined and could vary with particular pathogen, toxin,duration of exposure, linker pharmacokinetics, etc.

[0069] The universal immunogens and immunity linkers of the presentinvention are administered to individuals using any appropriate route.Appropriate routes of administration include, but are not limited to,oral, inhalation, parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraoccular, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, subcutaneous, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, transmucosal, intranasal,iontophoretic means, and transdermal means. Differing types of immuneresponse are sometimes triggered by different routes of administrationof an antigen, and the preferred route for the particular immuneresponse is known to those skilled in the art. The present invention isnot limited by the route of administration of the universal immunogen orimmunity linker.

[0070] With regard to the bacteriophage linker molecules andbacteriophage universal immunogens, both can be administered as thepurified phage or as a bacterial clone containing it. In a preferredembodiment, a lytic bacteriophage is administered to an individual as aportion of, or contained within, a bacteria. The bacteriophage can bedelivered by known administration methods that would allow for anoptimum response to the target.

[0071] The compositions described herein are also contemplated toinclude pharmaceutical compositions comprising immunity linkers oruniversal immunogens and at least one of any suitable auxiliary such as,but not limited to, diluent, binder, stabilizer, buffers, salts,lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Examples andmethods of preparing such sterile solutions are well known in the artand can be found in well known texts such as, but not limited to,REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, Ed., 18th Edition, MackPublishing Co. (1990)). Pharmaceutically acceptable carriers can beroutinely selected that are suitable for the mode of administration,solubility and/or stability of the compound. Pharmaceutical excipientsand additives useful in the present invention include, but are notlimited to, proteins, peptides, amino acids, lipids, and carbohydrates.The pharmaceutical compositions comprising the compounds of the presentinvention can also include a buffer or a pH adjusting agent.Additionally, pharmaceutical compositions of the invention can includepolymeric excipients/additives.

[0072] The term “adjuvant” as used herein is any substance whoseadmixture with the universal immunogen increases or otherwise modifiesthe immune response generated thereby. Any adjuvant system known in theart can be used in the composition of the present invention. Suchadjuvants include, but are not limited to, Freund's incomplete adjuvant,Freund's complete adjuvant, polydispersed β-(1,4) linked acetylatedmannan (“Acemannan”), Titermax® (polyoxyethylene-polyoxypropylenecopolymer adjuvants from CytRx Corporation), modified lipid adjuvantsfrom Chiron Corporation, saponin derivative adjuvants from CambridgeBiotech, killed Bordatella pertussis, the lipopolysaccharide (LPS) ofgram-negative bacteria, large polymeric anions such as dextran sulfate,and inorganic gels such as alum, aluminum hydroxide, or aluminumphosphate.

[0073] For oral administration, pharmaceutical compositions can be inthe form of a tablet or capsule, such as discrete units such ascapsules, cachets or tablets each containing a predetermined amount ofthe immunity linkers; as a powder or granules; as a solution or asuspension in an aqueous liquid or a non-aqueous liquid; or as anoil-in-water liquid emulsion or a water-in-oil emulsion and as a bolus,etc. A tablet may be made by compression or molding, optionally with oneor more accessory ingredients. The tablets may be optionally coated orscored and may be formulated so as to provide a slow or controlledrelease of the active ingredient therein. In one embodiment, theimmunity linker or universal immunogen is provided by orallyadministering E. coli infected with a bacteriophage immunity linker orbacteriophage universal immunogen.

[0074] In addition, the compositions of the present invention may beincorporated into biodegradable polymers allowing for sustained releaseof the immunity linkers, for example, the polymers being implanted forslow release of the immunity linkers. Biodegradable polymers and theiruses are described, for example, in detail in Brem et al., 74 J.NEUROSURG. 441-46 (1991).

[0075] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes that render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

[0076] Formulations suitable for topical administration in the mouthinclude lozenges comprising the ingredients in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the immunity linkers or universalimmunogens to be administered in a suitable liquid carrier. The liquidforms may include suitably flavored suspending or dispersing agents suchas the synthetic and natural gums, for example, tragacanth, acacia,methyl-cellulose and the like. Formulations for rectal administrationmay be presented as a suppository with a suitable base comprising, forexample, cocoa butter or a salicylate. Formulations suitable for vaginaladministration may be presented as pessaries, tamports, creams, gels,pastes, foams or spray formulations containing in addition to the activeingredient such carriers as are known in the art to be appropriate.

[0077] The compositions of the present invention may also be entrappedin microcapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nanoparticles andnanocapsules) or in macroemulsions. REMINGTON'S PHARMACEUTICAL SCIENCES(A. Osol ed., 16th ed. (1980)).

[0078] The present invention provides stable formulations as well aspreserved solutions and formulations containing a preservative as wellas multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising the immunity linker compositions disclosedherein in a pharmaceutically acceptable formulation.

[0079] In general, the compositions disclosed herein may be used aloneor in concert with therapeutic agents at appropriate dosages defined byroutine testing in order to obtain optimal efficacy while minimizing anypotential toxicity. The dosage regimen utilizing a composition of thepresent invention may be selected in accordance with a variety offactors including type, species, age, weight, sex, medical condition ofthe patient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular composition or therapeutic agent employed. A physician orveterinarian of ordinary skill can readily determine and prescribe theeffective amount of the immunity linker and/or universal immunogenrequired to prevent, counter, or arrest the progress of the condition.

[0080] The dosages of a composition disclosed herein may be adjustedwhen combined to achieve desired effects. Methods are known in the artfor determining effective doses for therapeutic and prophylacticpurposes for the disclosed pharmaceutical compositions. Morespecifically, the pharmaceutical compositions may be administered in asingle dose, or a single daily dose or the total daily dosage may beadministered in divided doses of two, three, or four times daily. Thedosage of the compositions may be varied over a wide range from about0.0001 to about 1,000 mg per individual or until an effective responseis achieved. The range may more particularly be from about 0.001 mg/kgto 10 mg/kg of body weight, about 0.1-100 mg, about 1.0-50 mg or about1.0-20 mg, for adults (at about 60 kg). The compositions may beadministered on a regimen of about 1 to about 10 times per day, for oneor multiple days, or once a week or once a month, or until an effectiveresponse is achieved. The pharmaceutical compositions of the presentinvention may be administered at least once a week over the course ofseveral weeks or months. Data obtained from cell culture assays andanimal studies may be used in formulating a range of dosages for use inhumans.

[0081] In addition, co-administration or sequential administration ofthe compositions of the present invention and other therapeutic agentsmay be desirable. A composition described herein can be administeredduring, before or after administration of any other therapeutic agent.

Methods of Production

[0082] Immunity linkers can be made in many ways, several of which aredescribed herein and are not to be seen as limiting the methods ofmaking immunity linkers. The universal immunogen, or first binding site,can be physically linked or conjugated, such as with known chemicalconjugation methods or molecules, to a molecule or organism with the atleast one second binding site that binds the target. In anotherembodiment, the immunity linker can be produced or manufactured as asingle molecule containing the first and second binding sites. Theimmunity linker may also comprise an organism. In yet anotherembodiment, the immunity linker consists of two active binding sitesconnected together by a rigid or flexible spacer such as a doublehelical region of RNA or DNA. A function of the spacer is to hold thetwo ends of the linker together, while preventing them from interacting.

[0083] The first and second binding sites of the present invention maybe identified and isolated by any method. Methods for isolating bindingmoieties for a target or immune response component can be determinedusing methods such as those of Mario Geysen. Geysen, et al., PNAS 198481(13):3998-4002 and Geysen et al., J. Immunol. Methods, 1987, 102 (2)259-74 describe an early method of peptide synthesis and screening,using Geysen's pin apparatus. Improvements to the original method andapplications of the methods have been taught in many publications,including, but not limited to, publications such as Geysen, et al.,Chem. Biol. 1996, 3(8):679-88; Schultz et al., Biotechnol. Prog., 1996,12(6):729-43; Carter, J M, Methods Mol. Biol. 1994, 36:207-23 (GeysenPEPSCAN procedure); Int. J. Pept. Protein Res. 1993, 42(1):1-9; Wagneret al., Comb. Chem. High Throughput Screen 1998 1(3):143-153; Edmundson,et al., Proteins, 1993, 16(3):246-67; Alexander et al., PNAS 199289(8):3352-6; Edmundson, et al., Ciba Found. Sump 1991, 158:213-25;Rodda et al., Australas Biotechnol. 1993, 3(6) 346-7; Tribbeck et al.,J. Immunol. Methods 1991, 139(2):155-66; Smith, G. P., Curr. Opin.Biotechnol. 1991, 668-73.

[0084] These combinatorial synthesis methods can be used to rapidlydetermine binding peptides that function as second binding sites inbinding the target. Randomly generated peptides can also be rapidlytested for binding to the target and provide second binding sites forbinding to the target. These peptides, protein fragments or peptides canbe conjugated to the first binding site to form an immunity linker.

[0085] In several embodiments of the present invention, the universalimmunogen comprises a first bacteriophage expressing a firstpolypeptide, and the immunity linker comprises a second bacteriophageexpressing both the first polypeptide and a second polypeptide. In someembodiments, the first bacteriophage is a wild-type form of abacteriophage and the second bacteriophage is a mutant, or recombinant,form of the same bacteriophage. Phage display techniques can be used toselect for a bacteriophage expressing either or both the firstpolypeptide and the second polypeptide. More specifically, phage displayis a selection technique in which a peptide or protein is expressed as afusion with a coat protein of a bacteriophage, resulting in display ofthe fused protein on the exterior surface of the phage virion. Phagedisplay allows for the selection of a peptide displayed on the outsideof a bacteriophage that binds to a target. This peptide, or some part ofit, functions as the second binding site.

[0086] Methods for making the nucleic acid aptamers, which form oneembodiment of a first and/or second binding site are known in the artand are taught at least in several patents referenced above. In general,the methods comprise making a nucleic acid ligand for any desiredtarget. The methods involve selection from a mixture of nucleic acidcandidates and step-wise iteration of structural improvement using thesame general selection theme, to achieve virtually any desired criterionof binding affinity and selectivity. For example, the SELEX methodallows for isolation of a single sequence variant in a mixturecontaining at least 10¹⁴ sequence variants. Aptamers generated using theSELEX methods or improvements or other methods are then used as thesecond binding sites for immunity linkers. The aptamers to any targetcan be generated in hours or days, linked to the linker portion and thefirst binding site of the immunity linker, and provided for protectionof a population.

[0087] All publications and patents mentioned herein are incorporatedherein by reference for the purpose of describing and disclosing, forexample, the constructs and methodologies that are described in thepublications, which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

[0088] It should be understood, of course, that the foregoing relatesonly to preferred embodiments of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in thisdisclosure.

EXAMPLE 1 Administration of Haemophilus influenzae Type B ImmunityLinker to Neonatal Rats

[0089]Haemophilus influenzae Type B (Hib) is an encapsulated bacterialpathogen that causes serious invasive diseases, particularly in youngchildren and the immunocompromised. The protective immune response toHib is directed against epitopes of the capsular polysaccharide (PS). Itis known that passive administration of anti-capsular polysaccharideantibody to rats inoculated with 10-100 Hib organisms intraperitoneallyprior to infection will protect them against bacteremia/sepsis. Theprimary effector mode of protection against Hib is by thecomplement-dependent bactericidal activity of the anti-PS antibodies.

[0090] For purposes of the present invention, a human Fab fragmentspecific for the Hib capsular polysaccharide (PS) was cloned. While thisFab fragment uses the same heavy and light chain variable regions of“native” antibodies, it lacks the CH2 and CH3 domains of the IgG heavychain. Accordingly, this Fab fragment alone is unable to bind complementand manifest bactericidal/protective activity. The Fab fragment (Fab41)serves as the second binding site of the immunity linker. This Fabfragment was linked to a phenylarsonate hapten that serves as the firstbinding site of the immunity linker. The resulting immunity linker waslabeled Fab41-ARS.

[0091] Neonatal rats received a subcutaneous injection ofanti-phenylarsonate antibodies made by injecting phenylarsonated keyholelimpet protein into adult rats and affinity purifying the antibodiesproduced on a phenylarsonate column. Eighteen hours later, Hib organismswere administered intraperitoneally to the neonatal rats. Two hourslater, Fab41-ARS, the linker, was injected intraperitoneally into theneonatal rats. Eighteen to twenty-four hours later, blood from theneonatal rats was plated on chocolate agar and Hib colonies counted. Theresults are shown in Table 1 below. TABLE 1 Group Anti-ARS Fab41-ARS HibCFU/ml 1 — — >10⁶, >10⁶, >10⁶ 2 + (1.0 mg) — >10⁶, 10⁵, 4 × 10⁴ 3 + (0.1mg) — >10⁶, 1.3 × 10⁵, 5.5 × 10⁴ 4 — + (100 μg) >10⁶, >10⁶, >10⁶ 5 — +(10 μg) >10⁶, >10⁶, >10⁶ 6 — + (1.0 μg) >10⁶, >10⁶, >10⁶ 7 + (1.0 mg) +(100 μg) 2.2 × 10⁴, <20, <20 8 + (1.0 mg) + (10 μg) 4.5 × 10⁴, 1.1 ×10⁴, 4 × 10² 9 + (1.0 mg) + (1.0 μg) 4.5 × 10⁴, 4.4 × 10⁴, 1.3 × 10⁴10 + (0.1 mg) + (100 μg) <20, <20, <20 11 + (0.1 mg) + (10 μg) 1.0 ×10⁵, 2.1 × 10⁴ 12 + (0.1 mg) + (1.0 μg) 1.1 × 10⁵, 7.2 × 10⁴, 6.6 × 10⁴

EXAMPLE 2 Alpha-galactosyl Epitope Immunity Linker

[0092] Recombinant knock-out mice lackingalpha-1,3-galactosyltransferase and consequently have a B- and T-cellimmune response to its product, the alpha-1,3-galactosyl-galactose bondor alpha-galactosyl epitope, are administered an immunity linkercontaining an alpha-galactosyl epitope. The alpha-galactosyl epitope isdescribed in Galili, U. and Avila, J. L., Alpha-Gal and Anti-Gal,Subcellular Biochemistry, Vol. 32, 1999. The immunity linker comprisesGal(alpha 1,3) Gal(beta 1,4)-GlcNAc-R, where the R represents a humanFab fragment specific for the capsular polysaccharide of Haemophilusinfluenzae type b (Hib). Ten minutes later, the mice areintraperitoneally administered a significant live dosage of Hib. After24 hours, by plating their blood on chocolate agar, the number of cfu inthe blood of the experimental mice is compared to the same measure inmice that have received the pathogen but not the prior treatment withthe immunity linker. The treatment with the Fab fragment linked to thealpha-galactosyl epitope inhibits the bacteremia relative to mice whichhad not received the linker. At some amounts of linker, inhibition isdose dependent.

EXAMPLE 3 Development of Phage Display Immunity Linkers with Specificityfor Anthrax

[0093] 1. Using standard phage display techniques, such as that sold byNew England BioLabs, with random oligonucleotides coding for a largenumber of random peptides, isolate a recombinant bacteriophage thatdisplays a peptide that is specific for B. anthracis spores, or othertoxins, toxin components (such as PA) or antigens of B. anthracis.

[0094] 2. Demonstrate in vitro that the recombinant bacteriophage actsas an immunity linker by 1) the binding of antibodies to anon-recombinant form of the bacteriophage and to the recombinantbacteriophage and 2) the binding of the recombinant bacteriophage to theanthrax spores.

[0095] 3. Immunize a subject with the non-recombinant bacteriophage.This immunization occurs by injection or by inhalation.

[0096] 4. Expose the subject to a composition comprising the recombinantbacteriophage that expresses the anthrax spore binding peptide. Usinginhalation administrative routes, provide adequate amounts of thecomposition effective to prevent anthrax infection in the lungs of thesubject.

[0097] 5. Upon exposure of the subject to anthrax through inhalationmeans, the subject is protected from infection by inhalation-typeanthrax.

[0098] Such procedures could also be used to stop or inhibit cutaneousor gastrointestinal anthrax exposure.

What is claimed is:
 1. A method of increasing an immune response to atarget in an individual comprising, administering to the individual aneffective amount of a composition comprising one or more immunitylinkers, wherein the linker molecules comprise at least one firstbinding site and at least one second binding site, wherein the secondbinding site binds to the target, wherein the individual has apre-existing immune response to the first binding site, or animmunological equivalent thereof, and wherein the immune response isselected from a cellular immune response and a humoral immune response.2. The method of claim 1, wherein the pre-existing immune response isinduced by administering to the individual a universal immunogencomprising the first binding site.
 3. The method of claim 1, wherein thepre-existing immune response is induced by administering to theindividual a universal immunogen that is an immunological equivalent ofthe first binding site.
 4. The method of claim 1, wherein thepre-existing immune response exists in the individual withoutadministration of a universal immunogen.
 5. The method of claim 1,wherein the second binding site comprises an antibody or a fragmentthereof.
 6. The method of claim 1, wherein the second binding sitecomprises a Fab antibody fragment.
 7. The method of claim 1, wherein thetarget is a pathogen.
 8. The method of claim 1, wherein the immunitylinker comprises a first bacteriophage.
 9. The method of claim 8,wherein the first binding site comprises a first polypeptide expressedby the bacteriophage and wherein the second binding site comprises asecond polypeptide expressed by the bacteriophage.
 10. The method ofclaim 9, wherein the pre-existing immune response is induced byadministering to the individual a universal immunogen comprising asecond bacteriophage that expresses the first polypeptide.
 11. Themethod of claim 10, wherein the first bacteriophage and/or the secondbacteriophage are each contained within one or more bacteria.
 12. Themethod of claim 1, wherein the individual is a human and the firstbinding site comprises an alpha-galactosyl epitope.
 13. The method ofclaim 1, wherein the individual is unable to mount an effective immuneresponse to the target prior to administration of the immunity linker.14. The method of claim 1, wherein the immune response is a cellularimmune response.
 15. The method of claim 1, wherein the immune responseis a humoral immune response.
 16. The method of claim 1, wherein thecomposition comprises a population of different immunity linkers whereinthe first binding sites differ in a) their specificity for differentepitopes on the immune response component, or b) their affinity for thesame epitopes on the immune response component.
 17. The method of claim16, wherein the immune response component comprises an antibody.
 18. Themethod of claim 1, wherein the composition comprises a population ofdifferent immunity linkers comprising second binding sites that differin a) their specificity for different epitopes on the target, or b)their affinity for the same epitope on the target.
 19. The method ofclaim 18, wherein the second binding sites each comprise an antibody ora fragment thereof.
 20. A method of increasing a humoral immune responseto a target in an individual comprising, administering to the individualan effective amount of a composition comprising one or more immunitylinkers, wherein the linkers comprise at least one first binding siteand at least one second binding site, wherein the first binding sitebinds to a humoral immune response component, or an immunologicalequivalent thereof, wherein the second binding site binds to the target,and wherein the target normally elicits a cellular immune response inthat or another individual.